Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization of the enzyme.
The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems.
Heimo
H
H
Department of Biochemistry and Food Chemistry, University of Turku, Turku, FIN-20014, Finland. heihei@utu.fi
Palmu
K
K
Suominen
I
I
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D000469:Alkaline Phosphatase